MECHANISMS OF SUPPRESSING CAMPTOTHECIN TOXICITY

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DESCRIPTION: (Applicant's Abstract) In recent years, DNA topoisomerase I (Top1) has emerged as the cellular target of an increasing number of antitumor agents. As exemplified by camptothecin (Cpt), these drugs target Top1 by increasing the stability of the covalent enzyme-DNA intermediate. During S-phase, the collision of advancing replication forks with Cpt-enzyme-DNA complexes results in the formation of DNA lesions that signal cell cycle arrest and cell death. Studies in yeast and mammalian cells indicate that overexpression of top1 is inherently cytotoxic, while mutations in Top1 that stabilize the covalent complex recapitulate the phenotypic consequences of Cpt treatment. Taken together, these data support a model where increased concentrations of covalent complexes, as a consequence of drug action, enzyme concentration or mutation, result in the production of potentially lethal DNA lesions. However, little is known about the nature of the lesions produced and the repair processes required for their resolution. Less clear is the fidelity of repair processes or the potential induction of secondary malignancies following the therapeutic application of Top1 poisons. This application aims to fill the void in our understanding of the cytotoxic action of Top1 poisons by investigating the cellular processes involved in suppressing the cytotoxic action of Cpt and the potential for DNA lesions induced by Top1 poisons to promote tumor formation. In yeast, overexpression of the ubiquitin isopeptidase, Ubp11, suppresses the cytotoxic action of Cpt, with little effect on Top1 activity. Further characterization of Ubp11 function will define the cellular processes involved in the formation and repair of DNA lesions induced by Cpt. Top1 mutants demonstrating distinct mechanisms of Top1 poisoning will be used to investigate the cytotoxic and mutagenic potential of different DNA lesions. The ability of Ubp11 (or human homologues) to suppress the action of these Top1 poisons will also be investigated, along with the consequences of yeast and mammalian cell survival on mutation rates and cell proliferation. To define the oncogenic potential of Top1-DNA lesions, Top1-induced neoplastic transformation will be assessed in untransformed fibroblasts and in a transgenic mouse model. These studies will provide insight into the ability of Top1-targeted drugs to induce tumors in pediatric and adult populations.